The long-term goal of this work is to elucidate the molecular mechanisms responsible for the altered retinal vascular function associated with diabetic retinopathy., the leading cause of adult blindness. We propose the following hypothesis: the loss of retinal auto regulation observed in diabetic retinopathy is due to an early phenotypic switch in the retinal arteries whereby increased levels of inducible nitric oxide synthetase (iNOS) replace the constitutively expressed, calcium- regulated endothelial nitric oxide synthase (eNOS). This calcium- independent increase in iNOS results in increased and continuous generation of nitric oxide (NO) and vasodilation. In healthy vessels and early on in diabetic vessels, free radical scavenging systems eliminate glucose-induced, oxygen-derived free radicals. However, if antioxidant defense systems fail, oxygen-derived free radicals combine with NO to form the toxic peroxynitrite, resulting in retinal vessel vasoconstriction. We will use a unique combination of experimental approaches to integrate information obtained at both the subcellular and cellular levels in single endothelial cells with observations and measurements made in intact small, pressurized retinal arteries from diabetic rats. Our main experimental tools will be rat and human endothelial cell cultures, fluorescence spectroscopy and electron spin trap resonance spectroscopy (for measurement of NO and reactive oxygen species), and immunohistochemistry. Advanced electrophysiology and imaging methodologies will be used to measure redox state in living cells and intact vessel function. We put forth 3 Specific Aims to test our hypothesis. In AIM 1 (cell culture studies), we will test the prediction that the cellular redox state and the availability of substrate and co- factors regulate the amount and the type of reactive oxygen formed by NOS in endothelial cells. In AIM 2 )studies using intact vessel preparations) we will examine the changes in vascular reactivity in intact pressurized retinal vessels from rats with type II diabetes in relation to increased iNOS activity and peroxynitrite formation. We will carefully map the initial and progressive changes in vascular reactivity in these animals by quantifying the "time course of change" in vascular reactivity and oxidative stress in vessels of these rats and then correlate these changes to those observ4ed in diabetic retinal arteries. In AIM 3 (whole animal studies), we will test whether mitigating the effects of iNOS in the BBZ/Wor diabetic rat improves blood retinal barrier and vascular dysfunction. A reduction in iNOS bioactivity will be accomplished either by cleaving iNOS mRNA using a ribozyme or by reducing enzymatic activity with pharmacological inhibitors. Results from these studies should suggest new therapeutic interventions for prevention of diabetic retinopathy that lead towards restoration of both retinal cell redox balance and retinal vascular function.